1. Field of the invention
The present invention relates to a scanning laser microscope which is extremely simple to adjust and combines confocal effects with detection sensitivity.
2. Description of the prior art
Research and development of scanning laser microscopes which use a laser beam as the scanning microscope light source has advanced quickly, and scanning laser microscopes are already in commercial use.
An example of the conventional scanning laser microscope is described below with reference to accompanying figures. A good example of the conventional scanning laser microscope is described in the Journal of the Japan Society for Precision Engineering (p. 35, July, 1991), and the construction is shown in FIG. 7.
This scanning laser microscope system uses three laser beams, a red laser beam 1R emitted from a He--Ne laser or other red laser source, a green laser beam 1G emitted from an Ar laser (515 nm) or other green laser source, and a blue laser beam 1B emitted from an Ar laser (488 nm) or other blue laser source. The other essential components are concave lenses 2R, 2G, and 2B, convex lenses 3R, 3G, and 3B, dichroic mirrors 4G, 4B, and 8G, 8B, a beam splitter 5, an objective lens 6, focusing lenses 9R, 9G, and 9B, pinholes 10R, 10G and 10B, detectors 11R, 11G, and 11B, video amplifiers 12R, 12G, and 12B, and a color monitor 13. The specimen being analyzed is shown as 7 in the figure.
As shown in FIG. 7, the red laser beam 1R is expanded by the concave lens 2R and convex lens 3R, passes through the dichroic mirrors 4G and 4B and the beam splitter 5, and is focused on the specimen 7 by the objective lens 6. The reflected light from the specimen 7 is collimated by the objective lens 6, reflected by the beam splitter 5 and passes the dichroic mirrors 8B, 8G, and is focused by the focusing lens 9R on the pinhole 10R. The light passing the pinhole 10R is detected by the detector 11R, and the output signal is amplified by the video amplifier 12R.
The green laser beam 1G (blue laser beam 1B) is expanded by the concave lens 2G (2B) and convex lens 3G (3B), is reflected by the dichroic mirror 4G (4B) and passes the beam splitter 5, and is focused on the specimen 7 by the objective lens 6. The reflected light from the specimen 7 is collimated by the objective lens 6, reflected by the beam splitter 5 to the dichroic mirrors 8G (8B), and reflected by the dichroic mirrors 8G (8B) to the focusing lens 9G (9B). The light is then focused by the focusing lens 9G (9B) on the pinhole 10G (10B). The passed light is detected by the detector 11G (11B), and the output signal is amplified by the video amplifier 12G (12B).
The video signal from the specimen 7 is obtained on the color monitor 13 by capturing these amplified signals and either fixing the light beam and scanning the specimen 7, or fixing the specimen 7 and scanning the light beam.
FIG. 8 illustrates the effect of inserting a pinhole. When the spot of the laser beam on the specimen is at objective lens focal point F, the return light passing through the objective lens 6 and the focusing lens 9 is refocused at the pinhole 10 as indicated by the solid line in the figure. The focused beam thus passes through the pinhole 10 and is incident upon the detector 11. When the spot is offset from focal point F to F', however, the return light is blocked by the pinhole 10 and is therefore not incident upon the detector 11 as shown by the dotted line.
A scanning laser microscope that uses pinholes as described is known as a "confocal" device. The advantages of the confocal microscope include an approximately 40 percent increase in resolution compared with non-confocal designs, extremely high contrast due to the cut-off of extraneous scattered light by the pinholes, and extremely high resolution in the depth of field. These effects are collectively called "confocal effects," and become more pronounced as the pinhole diameter decreases.
The conventional confocal scanning laser microscope as thus described does present the following problems, however. Specifically, because the focused spot on the detector side in this conventional device has a three-dimensional spread, pinhole position adjustment also requires adjusting the position on three axes. In addition, adjusting the position of the pinhole must also be on the order of the laser beam wavelength because the pinhole 10 diameter is approximately the circular area of the focused spot, that is, on the order of the wavelength.
It is therefore extremely difficult to adjust the pinhole, and the position is also easily affected by natural offsets in the optical system due to secular change. In addition, while the confocal effects can be improved by decreasing the pinhole diameter, this also decreases the amount of light incident on the detector. As such, there is an obvious trade-off between confocal effects and detection sensitivity.